Induction and suppression of tick cell antiviral RNAi responses by tick-borne flaviviruses.

Esther Schnettler,Libor Grubhoffer,Richard J Kuhn,Sabine Weisheit,Gorben P Pijlman,Lesley Bell-Sakyi,Samuel H Lewis,Gerald Barry,Mayuri Sharma,Ernest A Gould,Mark G Sterken,Alain Kohl,Darren J Obbard,Melanie Mcfarlane,Hana Tykalová,John K Fazakerley,Sonja M Best,Margo E Chase-Topping,Mick Watson

doi:10.1093/nar/gku657

Abstract

Arboviruses are transmitted by distantly related arthropod vectors such as mosquitoes (class Insecta) and ticks (class Arachnida). RNA interference (RNAi) is the major antiviral mechanism in arthropods against arboviruses. Unlike in mosquitoes, tick antiviral RNAi is not understood, although this information is important to compare arbovirus/host interactions in different classes of arbovirus vectos. Using an Ixodes scapularis-derived cell line, key Argonaute proteins involved in RNAi and the response against tick-borne Langat virus (Flaviviridae) replication were identified and phylogenetic relationships characterized. Analysis of small RNAs in infected cells showed the production of virus-derived small interfering RNAs (viRNAs), which are key molecules of the antiviral RNAi response. Importantly, viRNAs were longer (22 nucleotides) than those from other arbovirus vectors and mapped at highest frequency to the termini of the viral genome, as opposed to mosquito-borne flaviviruses. Moreover, tick-borne flaviviruses expressed subgenomic flavivirus RNAs that interfere with tick RNAi. Our results characterize the antiviral RNAi response in tick cells including phylogenetic analysis of genes encoding antiviral proteins, and viral interference with this pathway. This shows important differences in antiviral RNAi between the two major classes of arbovirus vectors, and our data broadens our understanding of arthropod antiviral RNAi.

Highlights

Tick-borne arboviruses of the Flaviviridae family are highly relevant to public health [1]
We identify and characterize key RNA interference (RNAi) players of the Ago family that interfere with Langat virus (LGTV) replication and describe characteristics of virusderived small interfering RNAs (viRNAs) in tick vector cells, which are different to viRNAs in mosquitoes
Reduced silencing was observed in cells expressing 3 untranslated RNA regions (UTRs) constructs compared to Maltose binding protein (MBP)-hepatitis delta virus ribozyme (HDVr) (Figure 5C). These results indicate that the 3 UTRs of LGTV and tick-borne encephalitis virus (TBEV) are able to interfere with the tick siRNA pathway

Summary

INTRODUCTION

Tick-borne arboviruses of the Flaviviridae family are highly relevant to public health [1]. Sequence analysis has identified putative Ago and Dcr genes in the I. scapularis genome [19] It is not known if these are transcribed and involved in tick antiviral RNAi responses. All studied insect specific viruses and plant-infecting viruses have been shown to express RNA silencing suppressor (RSS) proteins which interfere with the RNAi response [20]. No RSS proteins have been identified for arboviruses evasion strategies have been suggested for the alphavirus Semliki Forest virus (SFV) [21], and the production of a subgenomic flavivirus RNA (sfRNA) interfering with the RNAi response was reported for mosquito-borne flaviviruses [22]. We identify and characterize key RNAi players of the Ago family that interfere with LGTV replication and describe characteristics of viRNAs in tick vector cells, which are different to viRNAs in mosquitoes. The results imply that the antiviral RNAi system in ticks is more complex and has important differences to that of mosquitoes

MATERIALS AND METHODS

RESULTS

Findings

DISCUSSION